Analysis of Pectin-derived Monosaccharides from Arabidopsis Using GC–MS

Pectin is a complex polysaccharide present in the plant cell wall, whose composition is constantly remodelled to adapt to environmental or developmental changes. Mutants with altered pectin composition have been reported to exhibit altered stress or pathogen resistance. Understanding the link between mutant phenotypes and their pectin composition requires robust analytical methods to detect changes in the relative monosaccharide composition. Here, we describe a quick and efficient gas chromatography–mass spectrometry (GC–MS)-based method that allows the differential analysis of pectin monosaccharide composition in plants under different conditions or between mutant plants and their respective wild types. Pectin is extracted from seed mucilage or from the alcohol-insoluble residue prepared from leaves or other organs and is subsequently hydrolysed with trifluoracetic acid. The resulting acidic and neutral monosaccharides are then derivatised and measured simultaneously by GC–MS. Key features Comparative analysis of monosaccharide content in Arabidopsis-derived pectin between different genotypes or different treatments. Procedures for two sources of pectin are shown: seed coat mucilage and alcohol-insoluble residue. Allows quick analyses of neutral and acidic monosaccharides simultaneously. Graphical overview

This protocol is used in: Plant Physiol.(2022), DOI: 10.1093/plphys/kiac165 Pectin is a complex polysaccharide present in the plant cell wall, whose composition is constantly remodelled to adapt to environmental or developmental changes.Mutants with altered pectin composition have been reported to exhibit altered stress or pathogen resistance.Understanding the link between mutant phenotypes and their pectin composition requires robust analytical methods to detect changes in the relative monosaccharid e composition.Here, we describe a quick and efficient gas chromatography-mass spectrometry (GC-MS)-based method that allows the differential analysis of pectin monosaccharide composition in plants under different conditions or between mutant plants and their respective wild types.Pectin is extracted from seed mucilage or from the alcohol-insoluble residue prepared from leaves or other organs and is subsequently hydrolysed with trifluoracetic acid.The resulting acidic and neutral monosaccharides are then derivatised and measured simultaneously by GC-MS.

Background
Primary cell walls consist of up to 90% polysaccharides including cellulose, hemicelluloses, and pectin (Pettolino et al., 2012;Höfte and Voxeur, 2017).Of those, pectin is characterised by high amounts of D-galacturonic acid, which forms the backbone in the pectic polysaccharides homogalacturonan and rhamnogalacturonan II.In a third pectic polysaccharide, rhamnogalacturonan I, the backbone consists of disaccharide units of D-galacturonic acid linked to L-rhamnose (Atmodjo et al., 2013).Further mono-or oligosaccharide side chains are linked to the backbone, resulting in a chemically complex structure (Harholt et al., 2010;Atmodjo et al., 2013).Pectin is the most abundant component of primary cell walls of many plant species and performs diverse functional roles, including the response to infection by various pathogens (Shin et al., 2021).In this context, it was reported that an Arabidopsis double mutant line, disrupted in the two galacturonic acid-producing enzymes GLUCURONATE 4-EPIMERASE 1 and 6, contained less homogalacturonan and concomitantly showed higher susceptibility towards infection with the pathogens Pseudomonas syringae pv maculicola ES4326 and Botrytis cinerea isolates (Bethke et al., 2016).Also, modifications of the pectin backbone have been connected to plantpathogen interactions.In Arabidopsis, POWDERY MILDEW RESISTANT5 (PMR5) is responsible for the acetylation of galacturonic acid, and the pmr5 mutant has been shown to possess enhanced resistance to powdery mildew (Vogel et al., 2004).Furthermore, the bxl4 Arabidopsis mutant that lost enzyme activity of BETA-XYLOSIDOSE4 (BXL4) exhibited increased susceptibility to the fungal pathogen B. cinerea.BXL4 has been shown to possess activities on cell wall-derived arabinans and xyloses (Guzha et al., 2022).Given the reported effects of changes in pectin abundance and composition, fast and straightforward methods to compare the relative composition of pectin in plants under different conditions or of different genotypes are essential.Different methods for compositional analysis have been described so far, including colorimetric assays, gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography-based approaches (Pettolino et al., 2012;Biswal et al., 2017;Bethke and Glazebrook, 2019;Dean et al., 2019).Among those, GC-MS-based methods require analytes volatile enough for GC separation.Two main methods to obtain volatile analytes are described in the literature: reduction and acetylation to alditol acetates or acidic methanolysis of the sample followed by trimethylsilylation.The latter procedure offers the advantage of analysing neutral and acidic monosaccharides simultaneously; however, methanolysis is a time-consuming process with incubation times of 18 h (Biswal et al., 2017).We therefore intended to combine shorter sample processing times with the simultaneous study of neutral and acidic monosaccharides.We describe a GC-MS-based analysis of cell wall pectin extracted from cell wall samples as alcohol-insoluble residue (AIR) or seed coat mucilage by water extraction.Subsequently, extracted pectin is hydrolysed with trifluoracetic acid (TFA) that can be evaporated to leave the monosaccharide monomers.These are then derivatised with O-methylhydroxylamine hydrochloride (MOX) and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) to yield volatile analytes to be analysed by GC-MS.

A. Plant growth
For individual experimental setup, we recommend plants to be grown according to your usual procedure.Pots with the various genotypes (3-4 pots, each with five plants per genotype) should be randomised, placed in the same tray, and grown together under the same conditions.As an example, the procedure to enable comparative pectin analysis on alcohol-insoluble residue (AIR) derived from leaves, as used in Guzha et al. (2022), is shown below.1. Prepare semi-sterile soil by heating it in the oven for 8 h at 80 °C.2. Transfer soil to 10 cm pots, water, and place 6-10 Arabidopsis seeds in the four corners and the centre of the pots.Cover with plastic dome.3.After stratification at 4 °C for two days, transfer to growth cabinet with short-day conditions (8 h light and 16 h darkness) at 22 °C, a relative humidity of 65%, and a light intensity of 120-140 μmol m -2 •s -1 .Remove the plastic dome after 3-4 days.4. Grow plants for 7-10 days, then thin out excess seedlings to leave five plants per pot (one plant per position).5. Grow until plants reach the desired growth stage depending on your experimental setup, e.g., 6-week-old plants for analysis of leaf AIR.A detailed description on plant growth and seed harvest for mucilage analysis is given in Dean et al. (2019) (see General note 1).

B. Preparation of alcohol insoluble residue (AIR)
The method used in this part of the protocol is adapted from Bethke et al. (2016) and involves the following steps: 1. Flash-freeze the organs of interest (leaves, stems, roots, whole rosettes) in liquid nitrogen.Soil debris attached to roots or stems can be gently washed off using sterile distilled Milli -Q water.Note: To minimise starch contamination, leaf samples can be harvested after keeping the plants in the dark for 48 h, if the experimental setup allows (see General note 2). 2. Grind the frozen plant material using mortar and pestle or a ball mill.Keep the plant material frozen throughout grinding and make sure that the plant material is ground as fine as possible to minimize noncell wall contamination.Pause point: The ground plant material can be stored at -80 °C.3. Transfer up to 100 mg of ground plant material in a pre-cooled 2 mL microcentrifuge tube.Keep samples cooled before the addition of ethanol in the next step.4. Add 1.5 mL of 70% (v/v) ethanol and vortex thoroughly. 5. Centrifuge at 18,000× g for 10 min at room temperature and remove supernatant.6. Repeat steps B4 and B5 as described above.7. Add 1 mL of the solvent mixture chloroform/methanol 1:1 (v/v) and vortex thoroughly.Caution: Work with chloroform-containing solutions under a fume hood.
Published: Aug 20, 2023 8. Centrifuge at 18,000× g for 10 min at room temperature and remove supernatant.Caution: Removal of the chloroform-containing solution should be carried out under a fume hood.9. Repeat steps B7 and B8 two times as described above.After the final washing step, the plant material should have a grey to off-white colour, as all chlorophyll-containing fractions have been removed.Carry out additional washes with chloroform/methanol 1:1 (v/v) if the plant material is still green.10.Add 1 mL of acetone to the plant material and vortex thoroughly.11.Centrifuge at 18,000× g for 10 min at room temperature and remove supernatant.12. Air dry the resulting AIR at room temperature overnight.Keep samples in a dust-free environment to avoid contamination while drying.13.To avoid starch contamination, it is possible to carry out an α-amylase digest (see General note 2).

C. Extraction and hydrolysis of pectin
1. Pectin analysis from AIR a. Prepare at least four replicates for each genotype or growth condition.Weigh 2 mg of AIR for each replicate and place in a 2 mL microcentrifuge tube.Record the exact weight of AIR placed in the microcentrifuge tube.Use AIR from plants grown at the same time and under the same conditions.b.Add 1.4 mL of water to the AIR.c. Add two glass beads (5 mm diameter) and homogenise AIR further with a ball mill for 1.5 min at 30 Hz. d.Incubate samples for 2 h at 90 °C under constant vigorous shaking (speed #4 on HLC HTM 130).e. Remove samples from heating block and allow the debris to settle for 5 min.Do not centrifuge the samples.f.Transfer 1 mL of the supernatant to a DURAN ® glass culture tube with screw cap.g.Dry the samples under a stream of N2 gas at 50 °C.
Note: The drying step might take a while due to the aqueous nature of the sample.h.Add 300 μL of 2 M TFA to the dried samples.Caution: TFA is corrosive and attacks skin, eyes, and mucous membranes.Wear chemical-resistant gloves and safety goggles and work with TFA only under a fume hood.i. Hydrolyse the samples for 1 h at 121 °C in a heating block for DURAN glass tubes.j.After hydrolysis, evaporate the TFA under N2 stream.k.Add 500 μL of water and 100 μL of the internal standard 0.05 mg/mL allo-inositol to the dried hydrolysis products.Pause point: For long-term storage, dissolved hydrolysis products can be kept at -20 °C for several months.l.Sample preparation for subsequent measurements: dry 20 μL of the dissolved hydrolysis products under a stream of N2 gas.m.Derivatise the dried samples with 15 μL of MOX (30 mg/mL in anhydrous pyridine) at room temperature overnight.Use freshly prepared MOX (see General note 3).Wear chemical-resistant gloves and work with pyridine and MOX solution only under a fume hood.Use solvent-resistant tips for pipetting.n.After overnight derivatisation, add 30 μL of MSTFA and measure by GC-MS after at least 1 h incubation time at room temperature.Incubation time with MSTFA before GC-MS analysis should not exceed 6 h. 2. Pectin analysis from mucilage a. Prepare at least four replicates of each genotype.Weigh 5 mg of seeds for each replicate and transfer them to a 2 mL microcentrifuge tube.Record the exact mass of prepared seeds.b.Add 1.4 mL of water to the seeds and vortex for a few seconds.c.Place samples on a rotary shaker for 2 h to extract mucilage.d.Remove the samples from the shaker and allow the seeds to settle for 1-2 min.Do not centrifuge the samples.e. Transfer 1 mL of the supernatant to a DURAN glass tube.f.Dry the samples under a stream of N2 gas at 50 °C.

Published: Aug 20, 2023
Note: The drying step might take a while due to the aqueous nature of the sample.g.Add 300 μL of 2 M TFA to the dried samples.
Caution: TFA is corrosive and attacks skin, eyes, and mucous membranes.Wear chemical-resistant gloves and safety goggles and work with TFA only under a fume hood.h.Hydrolyse the samples for 1 h at 121 °C in a heating block for DURAN glass tubes.i.After hydrolysis, evaporate the TFA under N2 stream.j.Add 500 μL of water and 100 μL of the internal standard 0.05 mg/mL allo-inositol to the dried hydrolysis products.

D. GC-MS analysis
1. Preparation of standards for calibration a. From the stock solutions of the monosaccharides, prepare standard combinations of defined molar amounts by combining aliquots of dilutions from the respective stock solutions with the correct volume.(For example, for a mix of 1 nmol arabinose and 1 nmol galactose, combine 4 μL each of 1/1,000 dilutions of the stock solutions of arabinose and galactose.)Add 5 μL of 0.05 mg/mL alloinositol.
For less abundant pectic monosaccharides, prepare 3-5 different molar amount samples in the range of 1-5 nmol.For highly abundant pectic monosaccharides, prepare 3-5 different molar amount samples in the range of 20-100 nmol.Low or high abundance of the various monosaccharides depends on the sample; galacturonic acid will generally be highly abundant, whereas rhamnose is expected to be highly abundant in seed mucilage but less abundant in AIR from leaves.If the sample composition is unclear, it might be helpful to compare initial GC-MS results of a low and a high concentration of each standard with the sample result and then adjust the concentrations accordingly.You can combine different monosaccharide standards in the following combinations: 1. Xylose, fucose, mannose 2.
Rhamnose, galacturonic acid Prepare all standard combinations and different molar amounts in triplicates.b.Dry the prepared standard solutions under a stream of N2 gas.c.Derivatise the dried standards with 15 μL of MOX (30 mg/mL in anhydrous pyridine) at room temperature overnight.Use freshly prepared MOX solution (see General note 3).Wear chemicalresistant gloves and work with pyridine and MOX solution only under a fume hood.Use solventresistant tips for pipetting.d.After overnight derivatisation, add 30 μL of MSTFA and analyse on GC-MS after at least 1 h incubation time.Incubation time with MSTFA before GC-MS analysis should not exceed 6 h. 2. Monosaccharide analysis with GC-MS a. Inject 1 μL of the derivatised sample with a split value of 10.Helium is used as carrier gas at a flow rate of 1 mL/min.b.Use the following temperature gradient: 150 °C for 2 min, ramp to 250 °C at 5 K/min (20 min), ramp to 320 °C at 15 K/min (4.67 min), 320 °C for 3 min.The inlet temperature is set to 250 °C.

Data processing and peak integration
Perform peak assignment and integration according to your usual procedure.Here, we describe exemplarily data processing and peak integration with MSD ChemStation Data Analysis.a. Define compound list for the individual monosaccharides in the software MSD ChemStation Data Analysis.Use the GC-MS runs of the standards to determine retention times and characteristic fragment ions of the individual monosaccharides.Include the internal standard allo-inositol as additional compound.
Note: For most monosaccharides, two peaks will be detected (Figure 1), as derivatisation with MOX yields two stereoisomers (compare retention indices in Table 1).For quantification, select the analyte peak without interference from other monosaccharide-derived analyte peaks.If there is no interference for any analyte peak of an individual monosaccharide, select the largest one.The measured values, calculations, and calculated values for the second xylose peak of Figure 1 are shown below as an example (Table 2).With the LINEST function of Excel, the correlation between the concentration of the standards and the average normalised values was calculated, leading to the following parameters (Data analysis step A4): m = 0.27434; b = -0.07287.
For the sample shown in Figure 1

General notes and troubleshooting
1.For mucilage analysis, it is critical that the mother plants (wild type and mutants) are grown together at the same standard growth conditions.Water the plants until the final siliques turn yellow and begin to dry out.A detailed description of plant growth conditions for mucilage analysis can be found in Dean et al. (2019).Grow several independent seed sets (including wild type) and perform independent mucilage analyses to detect variations caused by slightly different growth conditions.2. Starch contamination in the sample will cause excessive glucose signals.For leaf samples, plastidial starch accumulation can be avoided by keeping the plants in the dark for 48 h before harvest.Alternatively, starch can be removed by an α-amylase digest followed by an ethanol extraction after AIR preparation (Pettolino et al., 2012).3. The derivatisation solution of 30 mg/mL MOX in anhydrous pyridine should not be older than three days to limit contaminations with water.4. Calibration values obtained from one set of standards can be used for an extended time, as long as the sample type stays the same.In our hands, calibration values could be used for ~100 sample analyses.
Pause point: For long-term storage, dissolved hydrolysis products can be kept at -20 °C for several months.k.Sample preparation for subsequent measurements: dry 20 μL of the dissolved hydrolysis products under a stream of N2 gas.l.Derivatise the dried samples with 15 μL of MOX (30 mg/mL in anhydrous pyridine) at temperature overnight.Use freshly prepared MOX (see Notes).Wear chemical-resistant gloves and work with pyridine and MOX solution only under a fume hood.Use solvent-resistant tips for pipetting.m.After overnight derivatisation, add 30 μL of MSTFA and measure by GC-MS after at least 1 h incubation time at room temperature.Incubation time with MSTFA before GC-MS analysis should not exceed 6 h.

Published: Aug 20
, 2023 c.The transfer line temperature is set at 280 °C; ionisation is done with an electron energy of 70 eV and an ion source temperature of 230 °C.Mass spectra are recorded in an m/z range of 40-500.

Figure 1 .
Figure 1.Example of the GC-MS chromatogram of monosaccharides from mucilage pectin as observed with MSD ChemStation Data Analysis.The vertical axis shows detected signals, while the horizontal axis shows the retention time in minutes after injection.allo-Inositol was used as internal standard (4).Fucose and glucuronic acid were not detected in this sample; the respective analyte peaks would have retention times of ca.8.9 and 9.1 min for fucose, and 12.8 and 13.6 min for glucuronic acid.The peak at 9.1 min does not have the correct mass spectrum when compared with an external fucose standard.

4 .
Normalise the obtained molar amount to the initial sample mass of AIR or seeds.You can use the normalised molar amounts to determine the relative composition of your pectin samples.Calculate average and standard deviations for the percentages of the different monosaccharide compounds.You can also normalise the contribution of the less abundant monosaccharides to one of the dominating monosaccharides (see for exampleGuzha et al., 2022).
, the following peak areas were integrated: Xylose peak 2: 4484042; allo-inositol: 100751631 This enabled the calculations shown below: Normalised value according to B2a: This protocol or parts of it have been used in the following research articles: Guzha et al. (2022).Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea (Figure 8E, 9F, S3A, S8, and S13).

Published: Aug 20, 2023
mg in 1 mL of water Note down the actual mass that you weighed in and adjust the volume using the following formula:

:
Pyridine is harmful if swallowed, in contact with skin, or if inhaled.Wear chemical-resistant gloves and work with pyridine and MOX solutions only under a fume hood.Use solvent-resistant tips for pipetting.

Table 1 . GC-MS detection of individual monosaccharides.
Possible target ions for quantification and the device-independent retention index are given in the middle columns.As an example, the retention times of the different monosaccharides as shown in Figure1are also given.Monosaccharide peaks that could not be detected in the respective sample are denoted as n.d.

Table 2 . Exemplary values for the calculation of molar amounts of xylose.
The first column highlights the concentrations used for the external standard of xylose, all prepared in triplicates.The second and third column s show the integrated peak values as obtained from MSD ChemStation Data Analysis for xylose and allo-inositol, respectively.For the normalised values (column four), the ratio of values for xylose to the values for alloinositol was calculated.In the lower section of the table, the averaged values for each concentration of the xylose standard are shown (Data analysis steps A1 and A2).